We seek to understand the molecular basis of early activation events in T-lymphocytes in response to physiologically important stimuli; the functional responses most important to our research are T-lymphocyte cytoskeletal reorganization, adhesion, migration, and gene transcription. We emphasize analysis of: 1) changes in serine/threonine phosphorylation as key signaling events which regulate these processes, and 2) the serine/threonine kinases that mediate such phosphorylation. One major project is to understand the spatial reorganization during chemokine-induced polarization of peripheral blood T cells (PBT) and to elucidate the biochemical basis for that reorganization using a combination of microscopic, biochemical and molecular genetic approaches. We are extending our analysis of the discovery described in the previous annual report, that chemokine rapidly induces moesin dephosphorylation in T-lymphocytes. Of particular importance are the findings that this dephosphorylation both: 1) facilitates disassembly of microvilli and 2) promotes lymphocyte polarization. Our studies of the signal transduction events underlying this process implicate small G-protein-mediated activation of phospholipase C. Protein phosphorylation is a central part of normal cell function as well as to carcinogenesis / metastasis. We have made major progress in characterizing fundamental processes that regulate kinase specificity, including: the precise peptide-specificity of AGC kinases, and the control of their recruitment. Despite hundreds of protein kinases in a cell, phosphorylation of any particular site is mediated by few kinases. Such specificity of phosphorylation is achieved in part by preferences of different kinases for different patterns of amino acids surrounding the phosphorylation site, i.e. peptide specificity. We have developed a new technique which appears to be of general applicability for determination of the detailed peptide specificity of a protein kinase, and for prediction of the likely sites in the proteome for phosphorylation by that kinase. We have used this approach to determine more accurately than previously possible the specificity of protein kinase C family members PKC-zeta and PKC-theta (and 5 mutants thereof). Comparison of predictions with measured phosphorylation by PKC revealed that our system successfully predicted 92% of peptide substrates with 91% specificity, which is much better than previous approaches. Among varied new findings, particularly notable is the importance of disfavored residues in determining peptide specificity. Disfavored residues contribute both to specificity between major kinase families, and amongst members of a subfamily. The foregoing determination of specificity is pertinent both to prediction of candidate phosphorylation sites in the proteome, and to prediction of which kinase is most likely to phosphorylate them. We are using these predictions, in concert with mass spectrometric and phospho-motif antibody approaches to identify physiologically relevant phosphorylation sites. Moreover, we have initiated studies of targeting with strategies for directing isolated kinase domains to rafts. Our data with other PKC isoforms indicates that most of the contribution of the regulatory domain of PKC-theta to theta's special ability to stimulate IL-2 transcription relates to theta's membrane targeting. We are now extending that to studies of other AGC kinases and are working towards a better understanding of the interplay between peptide specificity and targeting in determining physiological roles of AGC kinases.